WWP2 Antibody, Biotin conjugated

What is WWP2 and why is it significant in research?

WWP2 is an E3 ubiquitin ligase belonging to the NEDD4-like protein family, which plays critical roles in regulating transcription, embryonic stem-cell fate, cellular transport, and T-cell activation processes . Its significance stems from its ability to accept ubiquitin from E2 ubiquitin-conjugating enzymes and transfer it directly to target substrates. WWP2 is particularly important in cancer research because it physically interacts with PTEN (a frequently mutated tumor suppressor) and mediates its degradation through ubiquitylation-dependent pathways . Researchers investigating cellular apoptosis and tumorigenicity frequently target WWP2 as it has been shown to control these processes.

Methodologically, understanding WWP2's function requires examining both its catalytic activity and its interactions with substrates, necessitating specific antibodies for detection and isolation.

What are the key specifications of biotin-conjugated WWP2 antibodies?

Biotin-conjugated WWP2 antibodies typically feature the following specifications:

These antibodies target the WWP2 protein (UniProt ID: O00308; Gene ID: 11060), which has several variants resulting from alternative splicing .

How does biotin conjugation enhance WWP2 antibody applications?

Biotin conjugation provides several methodological advantages for WWP2 detection:

• Enhanced sensitivity: The biotin-streptavidin system offers one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), enabling highly sensitive detection even of low-abundance WWP2 protein .

• Signal amplification: Each biotin molecule can bind multiple streptavidin molecules, each carrying multiple reporter molecules, creating significant signal enhancement compared to conventional secondary antibody detection.

• Versatility in detection systems: Biotin-conjugated antibodies can be detected using various streptavidin-linked reporters (HRP, fluorophores, gold particles), making them suitable for diverse applications including Western blotting, immunohistochemistry, and ELISA .

• Reduced background in multi-staining protocols: Biotin-conjugated primary antibodies eliminate the need for species-specific secondary antibodies, reducing cross-reactivity in multi-antibody staining protocols.

When designing experiments, researchers should consider that endogenous biotin must be blocked in biotin-rich tissues to prevent non-specific background signals.

How should researchers optimize Western blotting protocols with biotin-conjugated WWP2 antibodies?

For optimal Western blot results with biotin-conjugated WWP2 antibodies:

Sample preparation:

• Use RIPA buffer supplemented with protease inhibitors and N-ethylmaleimide (10 mM) to prevent deubiquitination

• Load 20-40 μg of total protein per lane

• Run 8% SDS-PAGE gels for better separation (WWP2 is approximately 100 kDa)

Blocking and antibody incubation:

• Block with 5% BSA in TBST (avoid milk as it contains endogenous biotin)

• Use a 1:1000 dilution of biotin-conjugated WWP2 antibody

• Incubate overnight at 4°C with gentle agitation

Detection:

• Use streptavidin-HRP (1:5000-1:10000)

• Incorporate appropriate controls:

  • Positive control: Cell lines with known WWP2 expression (e.g., C2C12, C6)

  • Negative control: WWP2 knockdown/knockout samples

  • Specificity control: Pre-adsorption with immunizing peptide

Expected results should show a primary band at approximately 100 kDa, though multiple bands may appear due to WWP2 isoforms or post-translational modifications .

What approaches can be used to study WWP2-mediated ubiquitination processes?

To investigate WWP2-mediated ubiquitination:

In vitro ubiquitination assays:

• Purify components: E1, E2 (UbcH5b), recombinant WWP2, target substrate (e.g., PTEN), and ubiquitin

• Perform reactions in buffer containing ATP and monitor ubiquitination by Western blotting

• Include controls with catalytically inactive WWP2 mutant (C838A)

• Detect substrate ubiquitination using anti-ubiquitin antibodies

Cellular ubiquitination analysis:

• Co-transfect cells with target substrate, HA-tagged ubiquitin, and wild-type or mutant WWP2

• Treat cells with proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

• Immunoprecipitate the substrate and detect ubiquitination by anti-HA Western blotting

• Compare results between wild-type WWP2 and catalytically inactive WWP2

Advanced analytical methods:

• Mass spectrometry to identify specific ubiquitination sites

• Proximity ligation assays to detect WWP2-substrate interactions in situ

• Analysis of ubiquitin chain topology (K48 vs. K63 linkages) using linkage-specific antibodies

Published research demonstrates that WWP2 can polyubiquitinate POU5F1 through K63-linked conjugation and promote its proteasomal degradation in embryonic stem cells .

How can researchers investigate WWP2's interaction with PTEN using biotin-conjugated antibodies?

To study WWP2-PTEN interactions:

Co-immunoprecipitation (Co-IP):

• Lyse cells in non-denaturing buffer to preserve protein complexes

• Use biotin-conjugated WWP2 antibodies with streptavidin beads to pull down WWP2

• Detect associated PTEN by Western blotting

• Perform reciprocal IP with PTEN antibodies to confirm interaction

• Include negative controls (IgG or irrelevant antibody) and positive controls (known WWP2 interactors)

Research has shown that WWP2 physically interacts with PTEN and can be co-immunoprecipitated from cells . Studies have demonstrated that WWP2 mediates PTEN polyubiquitination and degradation, affecting cellular apoptosis and tumorigenicity .

In vitro binding assays:

• GST-pulldown assays using bacterially expressed glutathione S-transferase-tagged proteins

• Surface plasmon resonance to measure binding kinetics between purified WWP2 and PTEN

Advanced approaches:

• Proximity ligation assay to visualize and quantify WWP2-PTEN interactions in fixed cells

• FRET-based assays to monitor interactions in living cells

• Domain mapping using truncated constructs to identify interaction interfaces

How do the regulatory mechanisms of WWP2 differ from other NEDD4-family E3 ligases?

WWP2 exhibits distinct regulatory mechanisms compared to other NEDD4-family members:

Structural differences:

• WWP2 utilizes a 2,3-linker regulatory mechanism, while NEDD4-1 employs a 1,2-linker mechanism

• These differences affect how the catalytic HECT domain interacts with regulatory domains

• Studies show deletion of the 2,3-linker in WWP2 is activating, similar to 1,2-linker deletion in NEDD4-1

Activation mechanisms:

• WWP2 is activated by NDFIP1, which induces conformational changes exposing the catalytic exosite

• NDFIP1 binding to WWP2 WW domains exposes the WWP2 exosite by weakening the exosite's interaction with the 2,3-linker

• This activation mechanism causes a 20-fold increase in binding affinity for certain ubiquitin variants

Functional implications:

• These mechanistic differences result in distinct ubiquitination patterns and substrate preferences

• Autoubiquitination studies reveal that WWP2 primarily modifies residues on or near its WW domains

• Mutations that mimic activation (e.g., Y369E in WWP2) show intermediate behavior between wild-type and linker deletion mutants

Understanding these differences allows researchers to develop specific inhibitors targeting WWP2 while sparing other family members.

What approaches can resolve data inconsistencies when working with WWP2 antibodies?

When facing inconsistent results with WWP2 antibodies, consider:

Antibody validation strategies:

• Genetic validation: Test antibody in WWP2 knockout/knockdown systems

• Epitope mapping: Use antibodies targeting different WWP2 regions to confirm results

• Competitive blocking: Pre-incubate antibody with immunizing peptide to verify specificity

• Orthogonal methods: Correlate protein detection with mRNA expression data

Technical troubleshooting:

• For multiple bands in Western blots: Determine if they represent isoforms (WWP2-N, WWP2-C), post-translational modifications, or non-specific binding

• For variable IHC staining: Optimize antigen retrieval methods, use endogenous biotin blocking (crucial for biotin-conjugated antibodies), and ensure consistent fixation

• For inconsistent immunoprecipitation: Adjust lysis conditions to preserve protein complexes

Data reconciliation approaches:

• Integrate results from multiple antibodies targeting different epitopes

• Consider biological variables affecting WWP2 expression or localization

• Document experimental conditions thoroughly to identify potential variables

Researchers should carefully select appropriate controls based on their experimental questions and systems.

How can WWP2 antibodies be used to elucidate its role in cancer progression?

WWP2 antibodies enable several sophisticated approaches to study cancer progression:

Mechanistic studies:

• Analyze WWP2-mediated PTEN degradation in cancer cells through ubiquitination assays

• Examine WWP2's effect on POU5F1 levels in stem cell-like cancer populations

• Investigate WWP2's role in ubiquitinating and degrading RPB1, affecting transcriptional processes

Clinical correlations:

• Perform immunohistochemistry on tumor tissue microarrays to correlate WWP2 expression with:

  • Tumor stage and grade

  • Patient survival and treatment response

  • PTEN protein levels (potentially using multiplex IHC)

Drug development applications:

• Screen for WWP2 inhibitors using antibody-based detection systems

• Validate target engagement in drug-treated samples

• Monitor WWP2 activity in patient-derived xenografts during treatment

Research has established that WWP2 plays a crucial role in tumorigenicity through its regulation of PTEN and other substrates . The development of selective WWP2 inhibitors represents a promising therapeutic avenue for cancers with dysregulated WWP2 activity.

What controls are essential when using biotin-conjugated WWP2 antibodies?

Essential controls for biotin-conjugated WWP2 antibody experiments include:

Specificity controls:

• Genetic controls: WWP2 knockout/knockdown samples

• Peptide competition: Pre-incubation of antibody with immunizing peptide

• Isotype controls: Non-specific IgG from the same species (rabbit)

• Alternative antibody validation: Compare results with antibodies targeting different WWP2 regions

Technical controls for biotin-conjugation:

• Endogenous biotin blocking: Critical for biotin-rich tissues/cells

• Streptavidin-only control: To assess non-specific binding

• Titration series: To determine optimal antibody concentration

• Storage controls: Compare fresh vs. stored antibody performance

Positive reference controls:

• Cell lines with confirmed WWP2 expression (e.g., C2C12, C6)

• Recombinant WWP2 protein standards

• Tissues with known WWP2 expression patterns

Quality control testing has validated these antibodies in applications including Western blotting in C2C12 and C6 cell lysates, IHC in human kidney tissue, and affinity binding assays with recombinant WWP2 protein fragments (KD of 3.0 × 10-7) .

What factors contribute to background staining when using biotin-conjugated WWP2 antibodies?

Common sources of background with biotin-conjugated antibodies include:

Endogenous biotin interference:

• Tissues with high biotin content (kidney, liver, brain) may show non-specific staining

• Solution: Use avidin/biotin blocking kit before antibody incubation

• Alternative: Consider non-biotin detection systems for problematic tissues

Technical factors:

• Insufficient blocking: Extend blocking time or increase BSA concentration

• Excessive antibody concentration: Titrate to optimal dilution (typically 1:100 for IHC)

• Insufficient washing: Increase number and duration of wash steps

• Cross-reactivity: Validate antibody specificity in your specific tissue/cell type

Sample preparation issues:

• Overfixation: Masks epitopes and increases background

• Incomplete deparaffinization: Results in non-specific staining

• Endogenous peroxidase activity: Use appropriate quenching steps

Methodological solutions include using biotin-free detection systems, implementing stringent blocking protocols, and carefully titrating antibody concentrations for each application and tissue type.

How can WWP2 antibodies be applied in multi-omics research approaches?

Integrating WWP2 antibodies into multi-omics research frameworks allows for comprehensive analysis:

Proteomics integration:

• Immunoprecipitation with WWP2 antibodies followed by mass spectrometry to identify:

  • Novel WWP2 interacting partners

  • Post-translational modifications on WWP2

  • Ubiquitinated substrates in different cellular contexts

Functional genomics correlation:

• Combine CRISPR screens for WWP2-dependent phenotypes with antibody-based validation

• Correlate WWP2 protein levels/activity with transcriptomic changes

• Integrate ChIP-seq data to identify transcriptional networks affected by WWP2

Spatial biology applications:

• Multiplex immunofluorescence to examine WWP2 distribution relative to substrates

• Single-cell analysis correlating WWP2 levels with cellular phenotypes

• Tissue-specific interactome mapping using proximity labeling approaches

These integrated approaches provide deeper insights into WWP2 function than single-method studies, revealing context-dependent roles across different tissues and disease states.

What novel methodologies are being developed to study WWP2-mediated ubiquitination dynamics?

Emerging technologies for studying WWP2 ubiquitination dynamics include:

Live-cell imaging approaches:

• Fluorescent ubiquitin sensors to monitor WWP2 activity in real-time

• FRET-based reporters for substrate ubiquitination

• Optogenetic tools to activate/inhibit WWP2 with spatiotemporal precision

Advanced biochemical techniques:

• Ubiquitin chain-specific antibodies to distinguish different linkage types

• Engineered ubiquitin variants (UbVs) as probes for WWP2 conformational states

• Reconstituted ubiquitination systems with purified components for mechanistic studies

Structural biology integration:

• Hydrogen-deuterium exchange mass spectrometry to analyze WWP2 conformational changes

• Cryo-EM studies of WWP2 in different activation states

• Molecular dynamics simulations to predict regulatory mechanisms

These approaches enable researchers to move beyond static snapshots of WWP2 function to understand the dynamic regulation and substrate selectivity of this important E3 ligase.